(194g) Effect of Ionic Conditions on the Purification of Plasmid DNA Using Ultrafiltration Membranes

It is well known that the structure of plasmid DNA varies significantly with solution ionic strength due to intramolecular electrostatic interactions between the charged phosphate groups. The objective of this work was to examine the effect of ionic conditions on the transmission of different plasmid isoforms through small pore size ultrafiltration membranes, with the goal of identifying conditions that can be used to enhance the purification of the supercoiled plasmid DNA which is of interest in gene therapy applications and as DNA-based vaccines.

             Experimental were performed with 3.0 and 16 kbp plasmids using composite regenerated cellulose and polyethersulfone ultrafiltration membranes. The different isoforms were generated by enzymatic digestion of the supercoiled plasmid, with the identity of the isoforms confirmed by agarose gel electrophoresis. The sieving coefficient of the linear isoform was nearly independent of solution ionic strength, but increased significantly with increasing filtrate flux due to the elongation of the highly flexible plasmid in the converging flow field into the membrane pores.  In contrast, the transmission of the open-circular and supercoiled plasmids both increased with increasing NaCl concentration. Similar trends were observed using solutions of MgCl₂. The dependence on the ionic condition was more dramatic for supercoiled plasmid, mainly due to changes in the superhelix structure in varying salt environments. The separation between the supercoiled and linear isoforms was enhanced at low ionic strength due to the increase in retention of the supercoiled plasmid.  In contrast, the separation between the supercoiled and open-circular isoforms was enhanced at high ionic strength, with the supercoiled plasmid collected in the permeate. These results clearly demonstrate the potential for enhancing the performance of membrane systems for plasmid DNA separations by controlling the ionic conditions to modify the transmission of the different plasmid DNA isoforms.

Keywords:  DNA, ultrafiltration, ionic strength, isoforms, DNA deformation